isolation and antibiotic sensitivity of bacillus

Malaysian Journal of Microbiology, Vol 6(2) 2010, pp. 127-132

127

Isolation and antibiotic sensitivity of Bacillus thuringinesis strain from dump soil

Sarker, D., Roy, N. and Yeasmin, T.*

Department of Biochemistry and Molecular Biology Rajshahi University, Rajshahi-6205. Bangladesh.

E-mail: [email protected]

Received 29 June 2009; received in revised form 26 October 2009; accepted 14 November 2009

_______________________________________________________________________________________________

ABSTRACT Bacillus thuringiensis (or Bt) is a commonly used as a pesticide. B. thuringiensis is a naturally-occurring soil bacterium, also occurs naturally in the gut of caterpillars of various types of moths and butterflies, as well as on the dark surface of plants. The xylanase producing bacterial strains were isolated from dump soil. The strains were isolated on xylan agar media and screening was carried out by xylanolysis method. To test the sensitivity of the isolates, ten different antibiotics were used. The strains were tested for resistance to doxycyclin, erythromycin, chloramphenical, cephallaxin, kanamycin, ampicillin, steptomycin, vancomycin, amoxycillin and neomycin. The strains showed sensitive to doxycyclin, erythromycin, chloramphenical, cephallaxin, kanamycin, ampicillin, steptomycin and vancomycin and also showed

resistance to amoxycillin and neomycin, when tested by disc diffusion method on nutrient agar plate confirmed by antibiotic spread plate method. The inhibitory effect of B. thuringiensis strains against the test bacteria Bacillus subtilis, Sarcina lutca, Shigella dysenteriae, Shigella sonnei and Pseudomonus aeruginosa examined. It was found that, B. thuringiensis S1, B. thuringiensis S2 and B. thuringiensis S3 strains showed an inhibitory effect on all of the test bacteria. Keywords: antibiotics, sensitivity, resistant, Bacillus thuringiensis _______________________________________________________________________________________________ INTRODUCTION B. thuringiensis (Bt) is a Gram-positive, soil-dwelling, spore-forming, rod-shaped bacteria. It is approximately 1 µm in width and 5 µm in length (Madigan and Martinko, 2005; Sakai et al., 2007). It grows at body temperature and produces a diamond-shaped crystal from its crystal proteins (Cry proteins) and uses it to fend off insects, predators, and other pathogens (Jimenez-Juarez et al., 2007). Microorganism that produces chemicals toxic to insects (Schnepf et al.,1998; Whalon and Ganghey, 1998) common insect targets are "moths, mosquitoes, blackflies, beetles, hoppers, aphids, wasps and bees as well as nematodes (Vadlamudi et al., 1995; Dorsch et al., 2002; Zhang et al., 2005). Bacillus thuringiensis occurs naturally in the environment, isolated from soil, insect and plant surface (Meadows, 1993; Schnepf et al., 1998; Aslim et al., 2002). B. thuringiensis

has been implicated in burn wound

infections and food-poisoning (Jackson et al., 1995; Damgaard et al., 1997). Little interest was shown in antimicrobial susceptibility profiles

of Bacillus species until

very recently. This was due to a combination of reasons:

the low recognition of the ability of Bacillus species

(Turnbull et al., 2004). While many antibiotics are known to exist, efforts to

discover new antibiotics still continue. Therefore, many species such as Streptomyces, Bacillus and Penicillium have been studied continuously for their ability to produce antibiotics (Brock and Madigan, 1991). In addition, due to the fact that Bacillus species have produced antibiotics in

the soluble protein structure and that these antibiotics have been found to be cheaper and more effective in studies conducted to date, these microorganisms are preferable for commercial production. Currently, the target is to produce antibiotics such as polymyxin and bacitracin from Bacillus (Debavov, 1982; Priest, 1989). It was reported that members of the species Bacillus generally produced polypeptide type bacteriocines, and that these antibiotics generally affect Gram positive bacteria (Huck et al., 1991; Marahiel et al., 1993). The apparent increase of the occurrence of antibiotic resistance among bacteria during the past years and its possible implication in public health has led to an intensified surveillance of bacterial resistance in many countries.

While biomedical scientists are discovering newer and most potent anti-microbial drugs, the pathogenic bacteria with their demonstration to survive, are gaining resistance them in a curious way. In a sensitive bacterial population, there may be a small number of drug resistant bacteria which develop resistant spontaneously as a result of mutation. More frequently resistance is due to the presence of additional gene(s) as extra chromosomal DNA known as R-factors (plasmids) (Choudhury,1995).

The aim of this work was to investigate the changes occurring in the bacterial their possible relation to antibiotic susceptibility, resistance to antimicrobial agents, information on the frequency and distribution of multidrug resistance patterns and mproduction of bioactive compounds by strains.

*Corresponding author

Mal. J. Microbiol. Vol 6(2) 2010, pp. 127-132

128

The primary purpose of the study was to determine the

susceptibilities of these species to a set of antibiotics

selected to have the greatest guidance value to clinicians

encountering

possibly B. thuringiensis infections. In determining some features of the microorganisms, which play an important role in soil development and biological control, our aim is to provide information for further biotechnical studies. MATERIALS AND METHODS Bacterial strain For the screening of xylanase producing Bacillus thuringiensis strains, dump soil samples were collected from Alam nagar of Rangpur, Bangladesh. Media and culture conditions Nutrient agar media, Czapek-dox agar media and Yeast extract xylan agar were used as a soild medium throughout the work. Yeast extract xylan agar plates were used for the isolation and identification of the bacteria and the bacteria were cultured at 37 °C. Isolation and characterization of bacteria All the xylanase producing bacterial strains which were isolated by their growth on xylane agar media as clear zones and xylanolytic properties were characterized according to the biochemical tests described in the “Berger Manual of Determinative Bacteriology”. Finally, Bacillus thuringienesis strains were identified by 16S rRNA. Antibiotic resistance and sensitivity The antibiotic resistant Bacillus thuringiensis strains were isolated from the selected strains containing of xylanolytic activity using the disc diffusion method (Bauer et al., 1966). A 16 h broth cultures of the collected strains when grown at 37 °C and was spread on both nutrient agar plate using sterilized glass spreader. Then ampicillin (10

µg/disc), amoxycillin (10 µg/disc), chloramphenical (30 µg/

disc), kanamycin (30 µg/disc), cephallxin (30 µg/disc),

doxycycline (30 µg/disc), erythromycin (15 µg/disc),

neomycin (30 µg/disc) and steptomycin ( 10 µg/disc) antibiotics were distributed on plate and kept the plates at 4 °C for 4-6 h, so that the antibiotic can diffuse on the agar media.

The plates were then incubated at 37 °C for 16 h and the growth of the bacteria was observed. The presence of a clear zone around the disc was the index of sensitivity to the antibiotic. The test results of antibiotic sensitivity were determined according to the inhibition zone diameter (Barry, 1986; Çetin and Gŭrler, 1989). The absence of such a clear zone or the presence of some colonies within the clear zone indicated that the collected strains were resistant to that antibiotic. The drug resistance bacteria tested by disc diffusion methods were again confirmed by

spreading its culture on the selected antibiotic plates of different concentrations. The plates were then incubated at 37 °C and observed on next day. The clear plate indicated that the strains sensitive to this selective concentration and presence of colonies on the plate indicated that the strains were resistant to that selective concentration. Antibacterial activity The primary assay performed in vitro by disc diffusion assay (Bauer et al., 1966). Five pathogenic bacteria were selected for the antibacterial studies, three of which were Gram negative and the other two were Gram positive (Table 1). The test organisms were collected from the Institute of Biological Sciences (IBSc), University of Rajshahi, Bangladesh. Table 1: Bacteria used for antibacterial study

Serial No. Test Organisms Strain Number Gram positive 1. Bacillus subtilus QL-40 2. Sarcina lutca QL-166 Gram negative 3. Shigella dysenteriae AL-35587 4. Shigella sonnei AJ-8992

5. Pseudomonus aeruginosa

CRL

The test organisms were inoculated in the medium to test tube and were agitated to ensure uniform dispersion of the organism into the medium. The medium was poured into the sterile Petridis and agitated clockwise, anticlockwise, left to right, right to left for uniform dispersion. 2 mg of crude extract (B. thuringiensis) was dissolved in 1 mL of ethyl acetate extract that served as a stock solution. Filter paper discs were taken in a petridish and sterilized in

oven at 110 °C for 1 h. Then 15 µL and 100 µL of the stock solution were placed on the paper discs separately

by micropipette to obtain 30 µg/disc and 200 µg/disc

respectively. The discs were then air-dried. Here,

kanamycin (30 µg/disc) used as a standard discs. The sample impregnated discs and standard disc were placed gently on the solidified agar plates seeded with the test organisms to ensure contact with the medium. Then the plates were kept in a refrigerator at 4 °C for 4-6 h in order to provide sufficient time to diffuse the compounds into the medium.

After incubation, the antibacterial activities of the test samples were determined by measuring the radius of the inhibition zones (Barry, 1986; Reinheimer et al., 1990).

RESULTS Isolation of bacteria

Bacterial strains were isolated from dump soil, which was described in materials and methods. In a preliminary experiment of this research, three bacterial strains were


129

spread on the two different agar plates for isolation and rapid identification of the xylanase-producing bacteria and

Figure 1: Xylanolysis colonies on xylan agar plate are surrounded by a clear, colourless zone after 48 h incubation at 37 °C

xylanolytic properties. The plate was then incubated at 37 °C for 48 h. the colonies which formed clear zone on the xylan agar plates were picked, and then further purified by pure colonies producing bacteria on the xylan agar plate were shown in Figure 1. Antibiotic resistance and sensitivity test The three drug resistant strains were isolated by disc diffusion method on nutrient agar plates against some commonly used antibiotics to see the resistance pattern of these strains. All strains showed multiple resistance to the antibiotic used .Resistant and sensitive strains were separated according to their diameter of zone of inhibition produced around the antibiotic disc and growth type and number of colonies on clear zone and comparative study with control strains (sensitive). The result of drug resistance pattern on nutrient agar showed that the three strains were resistant to amoxycillin and neomycin. Whereas, sensitive to doxycyclin, erythromycin, chloramphenical, cephallaxin, kanamycin, ampicillin, steptomycin and vancomycin (Figure 2). The results of overall antibiotic sensitivity tests on the agar plates have shown in the Figure 3. Antibiotic spread plate method against different selection concentration of antibiotics Antibiotic resistance strains through the disc diffusion method were also reisolated and again the drug resistant were confirmed by antibiotic spread plate method with different selective antibiotic concentration (10, 20, 30,

50,100 µg/mL). The antibiotics used were amoxicillin and neomycin for confirmation of drug resistance of those strains was isolated from disc diffusion method. The bacterial samples of B. thuringiensis was remarked as highly resistant colonies on these antibiotic plates indicated that all these strains were resisstant to these selective concentrations of antibiotics. While no growth at next other concentrations indicated that these strains were

sensitive to these selective to these concentrations of antibiotic.

Figure 2: Antibiotic sensitivity and resistant pattern strain (S1) on xylan agar plate

Antibiotic sensitivity test on nutrient agar plate

0

5

10

15

20

25

30

35

40

45

Ap A CHO Cep Do E K N St Van

Antibiotics

Dia

mete

r o

f th

e c

lear

zo

ne

(mm

) S1

S2

S3

S1: Strain 1; S2:Strain 2; S3: Strain 3

Figure 3: Antibiotic sensitivity test on nutrient agar plate Antibacterial activity Generally, antibacterial screening is for primary selection of the compounds as therapeutic agent. The antibacterial screening is done by primary assay, a qualitative assay to determine whether the compounds are antibacterially

active or not. As shown in Table 2. A 30 µg/mL of the

ethyl acetate extract of the cultural broth of B. thuringiensis bacteria strains, showed antibacterial activity against the tested pathogenic bacteria. The maximum antibacterial activities were observed against Pseudomonas aeruginosa for the tested extract. The strains showed a relatively lower inhibitory effect on Shigella dysenteriae. DISCUSSION In this study, bacterial strains were isolated from dump soil, which degraded α-1,4 xylan and to belong B.

Ap = Ampicillin, E = Erythromycin A = Amoxycillin K = Kanamycin CHO = Chloramphenical N = Neomycin Cep = Cephallxin St = Steptomycin Do = Doxycycline Van = Vancomycin


130

thuringiensis. It was observed that, the strains were resistant to two antibiotics i.e. amoxicillin and neomycin (Figure 4). The antibiotic resistance in each of these three

strains was confirmed by antibiotic spread plate method using amoxycillin (0.5 mg/mL) and neomycin (0.5 mg/mL)

Table 2: Antibacterial activity of the ethyl extracts (30 µg disc-1

) of B. thuringiensis strains against standard kanamycin

(30 µg/disc)

S1=Strain 1, S2=Strain 2 and S3=Strain 3

Figure 4: Antibiotic resistance test on nutrient agar plate

in 10, 20, 30, 50 and 100 µg/mL concentration on nutrient

agar plates. Present investigation suggests that the ethyl acetate

extract of the cultural broth of B. thuringiensis bacteria (strain1) contained some antibacterial components, which have activity against both Gram positive and Gram-negative bacteria [Figure 5 (a, b, c, d and e)]. All of the B. thuringiensis

isolates were resistant to amoxicillin,

ampicillin, ceftriaxone,

penicillin and oxacillin while susceptible to the remaining

antimicrobials (Luna et al.,

2007). Xylanase producing Aeromonas strains were resistant to amoxycillin, ampicillin, cotrimazole, and sensitive to erythromycin, tetracycline and doxycyline (Roy and Abedin, 2002; Roy et al., 2003).

The high level of drug resistance in tested bacterial strains could be related to the production of antimicrobial compounds found in some fish-farm isolates belonging to the genus Bacillus. The antimicrobial activity and resistance to antimicrobials of bacteria from a sediment habitat can help explain the selection and persistence of such strains in this particular ecology (Chelossi et al., 2003).

S t ra ins o f B. thur ing iens is , B. sub t i l i s , B. stearothermophilus, B. liceniformis, B. megaterium and B. cereus have been reported to produce substances like bacteriocin. Bacteriocins are ribosomally synthesized antimicrobial peptides produced by a number of different bacteria (Parker and Collier, 1990). As for the inhibitor

Figure 4: Antibacterial activity of the ethyl acetate extracts

of strain S1 and standard kanamycin (30 µg/disc) effect of isolates; it was observed that B. thuringiensis D1, B. thuringiensis D3, had an inhibitory effect on all of the four test bacteria (Aslim et al., 2002).

B. thuringiensis showed an antimicrobial effect on gram (+) bacteria and B. thuringiensis produced thuricin. The Bacillus strains isolated from soil were tested on the antibiotics ampicillin, kanamycin, streptomycin, chloramphenicol, tetracycline, erythromycin, gentamicin and novobiocin proved to be resistant to four different antibiotics (Bernhard et al.,1987), it is recommended that this strain be used in certain biotechnological studies (Chelossi et al., 2003).

A large variety of specific biochemical functions, such as fertility, resistance to antimicrobial drugs, production of bacteriocins, and production of toxins, have been attributed to some plasmids (Cohen,1976; Bernhard et al.,1978).

Strain No. Bacillus subtilis

Sarcina lutca Shigella dysenteriae

Shigella sonnei Pseudomonas aeruginosa

S1 ( EA 30 µg/disc) 9±0.05 11±0.10 1±.05 4±.20 13±.30

S2( EA 30 µg/disc) 6±0.02 10±0.10 1±.05 3±.1 10±.20

S3( EA 30 µg/disc) 10±0.05 11±0.10 2±.05 5±.02 14±.30

e

a b

c d

Antibiotic resistance test on nutrient agar plate

0

5

10

15

20

25

10 20 30 50 100 10 20 30 50 100

Amoxycillin Neomycin

Antibiotic concentrations

Dia

mete

r of th

e c

lear

zone (m

m)

S1 S2 S3


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For the treatment of B. thuringiensis and other Bacillus

infections, there is little advice found for treatment.

Yet

various Bacillus species demonstrated small populations

with some form of resistance to clindamycin

and erythromycin. Therefore the wider choice of newer

antimicrobials can be useful in treating an infection.

In conclusion, this paper has broadened the number of antimicrobials

potentially useful against B. thuringiensis.

Sensitive to doxycyclin, erythromycin, chloramphenical, cephallaxin, kanamycin, ampicillin, steptomycin and vancomycin testing by

disc diffusion method.

Resistance

in these species to amoxycillin and neomycin was confirmed by antibiotic spread plate method. REFERENCES Aslim, B., Sağlam, N. and Beyatli, Y. (2002).

Determination of some properties of Bacillus isolated from soil. Turkish Journal of Biology 26, 41-48.

Barry, A. L. (1986). Procedure for Testing Antimicrobial Agent in Agar Media: Theoretical Considerations. Lorian V: Antibiotics in Laboratory Medicine, 2

nd edn.

Williams-Wilkins, Baltimore-London. Bauer, A. W., Kirby, W. M. M. and Sherris, J. C. (1966).

Antibiotic susceptibility testing by a standard single disk method. American Journal of Clinical Pathology 45, 493-496.

Bernhard, K., Schempf, H. and Goebel, W. (1978). Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis. Journal of Bacteriology 133, 897-903.

Brock, T. D. and Madigan, M. T. (1991). Biology of Microorganisms. 6

th edn. Prentice-Hall International

Inc., USA. Çetin, E. T. and Gŭrler, N. (1989). Antibiotic susceptibility

tests of bacteria. Kŭkem Dergisi 2, 97-105. Chelossi, E., Vezzull, L., Anna, M., Branzoni, M.,

Fabiano, M., Riccardi, G. and Banat, I. M. (2003). Antibiotic resistance of benthic bacteria in fish-farm and control sediments of the Western Mediterranean. Aquaculture 219, 1-15.

Choudhury, M. R. (1995). Modern Medical Microbiology. 5

th edn. Choudhury, Dhaka, Bangladesh. pp. 58.

Cohen, S. N. (1976). Transposable Elements and Plasmid Evolution. Nature. Press. London. pp. 263.

Damgaard, P. H., Granum, P. E. and Bresciani, J. (1997). Characterization of Bacillus thuringiensis isolated from infections in burn wounds. FEMS Immunology and Medical Microbiology 18, 47-53.

Debavov, V. G. (1982). The Industrial Use of Bacilli In: The Molecular Biology of Bacilli1. Dubnau,D. A.(eds.). Academic Press. FL, USA. pp. 331-333.

Dorsch, J. A., Candas, M., Griko, N. B., Maaty, W. S. A., Midboe, E. G., Vadlamudi, R. K. and Bulla, L. A. Jr. (2002). Cry1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of BT-R(1) in Manduca sexta: Involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis. Insect Biochemistry Molecular Biology 32, 1025-1036.

Huck, T. A., Porter, N. and Bushell, M. E. (1991). Positive selection of antibiotic producing soil isolates. Journal of General Microbiology 137, 2321-2329.

Jackson, S. G., Goodbrand, R. B. and Ahmed, R. (1995). Bacillus cereus and Bacillus thuringiensis isolated in a gastroenteritis outbreak investigation. Letters in Applied Microbiology 21, 103-105.

Jimenez-Juarez, N., Munoz-Garay, C., Gomez, I., Saab-Rincon, G., Damian-Alamazo, J. Y., Gill, S. S., Soberon, M. and Bravo, A. (2007). Bacillus thuringiensis Cry1Ab mutants affecting oligomer formation are non toxic to Manduca sexta larvae. The Journal of Biological Chemistry 282(29), 21222-21229.

Luna, A. V., King, S. D., Gulledge, J., Cannons, C. A., Amuso, T. P. and Cattani, J. (2007). Susceptibility of Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides and Bacillus thuringiensis to 24 antimicrobials using Sensititre

® automated

microbroth dilution and Etest® agar gradient diffusion

methods. Journal of Antimicrobial Chemotherapy 60(3), 555-567.

Madigan, M. and Martinko, J. (2005). Brock biology of microorganisms. 11

th edn. Prentice Hall, USA. pp.

545-572 Marahiel, M. A., Nakano, M. M. and Zabar, P. (1993).

Regulation of peptide antibiotic production in Bacillus. Molecular Microbiology 7, 631-636.

Meadows, M. P. (1993). Bacillus thuringiensis in the environment: Ecology and risk assessment. In: Bacillus thuringiensis. An environmental biopesticide: Theory and Practical. Enwistle, P. F., Cory, J. S., Bailey, M. J., Higgs, S. (eds.). Wiley West Sussex, UK. pp. 193-220.

Parker, M. T. and Collier, L. H. (1990). Principles of bacteriology, virology and immunity. 7

th edn. Vol. 2.

D. C. Decker, Philadelphia. Priest, F. G. (1989). Products and applications. In:

Bacillus biotechnology. Hand Book 2. Harwood, C.R. (eds.) Plenum. New York-London. pp. 293-320.

Reinheimer, J. A., Demkow, M. R. and Condioti, M. C. (1990). Inhibition of coliform bacteria by lactic cultures. Australian Journal of Dairy Technology 5-6.

Roy, N. and Abbedin, Z. M. (2002). Isolation and molecular characterization of xylanase–producing bacteria from drain water. Online Journal of Biological Sciences 2(6), 399-402.

Roy, N., Belauddin, M. and Shajahan, M. (2003). Studies on Multiple Antibiotic Resistance gene in Aeromonas . Biotechnology 2(2):101-108.

Sakai, H., Howlader, M.T., Ishida, Y., Nakaguchi, A.,

Oka, K., Ohbayashi, K., Yamagiwa, M. and Hayakawa, T. (2007). Flexibility and strictness in functional replacement of domain III of cry insecticidal proteins from Bacillus thuringiensis. Journal of Bioscience and Bioengineering. 103, 381-383.

Schnepf, E., Crickmore, N., VanRie, J., Lereclus, D., Daum., Feitelson, J., Zeigler, D. R., and Dean, D. H. B. (1998). Bacillus thuringiensis and its pesticidal


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crystal proteins. Microbiology and Molecular Biology Reviews 62, 775-805.

Turnbull, P.C., Sirianni, N.M., LeBron, C.I., Samaan, M.N., Sutton, F.N., Reyes, A.E., Peruski, L.F. Jr. (2004).MICs of selected antibiotics for Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides from a range of clinical and environmental sources as determined by the Etest. Journal of Clinical Microbiology 42(8), 3626-3634.

Vadlamudi, R., Weber, E., Inhae, J., Ji, T.H. and Bulla Jr., L.A. (1995). Cloning and expression of a receptor for an insecticidal toxin of Bacillus thuringiensis. The Journal of Biological Chemistry. 270, 5490-5494.

Whalon, M. E. and MaGanghey, W. H. (1998). Bacillus thuringiensis Use and Resistance Management. Insecticide with Novel Modes of action Mechanism and Application Ishaaya. I. Deheele. (eds.). Springer –Verlag. New York. pp. 106-137.

Zhang, X., Candas, M., Griko, N.B., Rose-Young, L. and Bulla, L. A. (2005). Cytotoxicity of Bacillus thuringiensis Cry1Ab toxin depends on specific binding of the toxin to the cadherin receptor BT-R(1) expressed in insect cells. Cell Death and Differentiation. 12, 1407-1416.

isolation and antibiotic sensitivity of bacillus

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